Project Summary/Abstract Addiction is a complex disorder involving interactions among a number of factors that influence drug use, including stress. Stress is a powerful, pervasive and unavoidable contributor to addiction and so understanding the neurobiological mechanisms responsible for development of addiction, and through which stress promotes drug use, remains a necessary area of study. A defining characteristic of addiction is loss of control over drug intake that, in the rat, is modeled by escalating patterns of drug self-administration (SA). In rats demonstrating stable cocaine SA, a stressor administered daily within the SA context, induces an escalation of drug use, suggesting that chronic stress influences the transition to an addicted phenotype. Stress-induced escalation is likely the result of long-lasting neuroadaptations and the proposed research seeks to identify neurobiological mechanisms that underlie the influence of stress on drug use. Stress-induced escalation of drug intake is glucocorticoid-dependent and likely involves neurobiological mediators that connect stress-responsive and reward systems in the brain, such as endocannabinoids (eCBs). In support of this, systemic administration of a cannabinoid receptor 1 (CB1R) antagonist attenuates cocaine intake and cocaine-seeking only in stress- escalated rats. Furthermore, intra-ventral tegmental area (VTA) administration of a CB1R antagonist attenuates cocaine intake identifying the VTA, a key reward-related brain region, as a critical site of eCB regulation. Importantly, eCB signaling in the VTA, through attenuation of inhibitory neurotransmission, is well positioned to regulate dopamine neuron activity and dopamine output to efferent brain regions such as the nucleus accumbens (NAc) shell. This proposal tests the novel hypothesis that repeated stress at the time of cocaine SA recruits the eCB system in the VTA to regulate dopamine neuron activity and output which ultimately results in expression of escalating patterns of cocaine use. In order to test this hypothesis, we will utilize behavioral, neurochemical, and neurophysiological techniques to assess the involvement of the eCB system in the influence of stress on cocaine use. First we will characterize the role of eCB signaling in stress-induced escalation of cocaine intake by examining the effects of pharmacological inhibition of CB1Rs in the VTA on escalated cocaine intake. Second, we will determine the role of eCB signaling in the VTA in regulation of downstream dopamine signaling by utilizing fast-scan cyclic voltammetry (FSCV) to examine changes in cocaine-induced phasic dopamine in the NAc shell in the presence or absence of CB1R antagonism in the VTA following stress-induced escalation of cocaine intake. Last, we will examine changes in eCB-mediated synaptic plasticity that are associated with stress- induced escalation of cocaine intake, through measurement of depolarization-induced and CB1R agonist- induced suppression of inhibition, using whole-cell patch clamp recordings in the VTA. These studies have treatment implications for subpopulations of addicts for whom stress is a contributing factor by identifying unique neurobiological mechanisms that may provide therapeutic targets.